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Insulin- and Glucose-Induced Phosphorylation of the Na⁺,K⁺-Adenosine Triphosphatase -Subunits in Rat Skeletal Muscle

Department of Clinical Physiology (A.V.C., J.W.R., H.W.-H., J.R.Z.), Karolinska Hospital, 171 76 Stockholm, Sweden; Department of Physiology and Pharmacology (A.V.C., J.W.R., E.F., H.W.-H., J.R.Z.), Karolinska Institute, 171 77 Stockholm, Sweden; Ludwig Institute for Cancer Research (M.V.K.), 751 24 Uppsala, Sweden; and Division de Néphrologie (E.F.), Hôpital Cantonal Universitaire, 1211 Genève 4, Switzerland

Address all correspondence and requests for reprints to: Alexander V. Chibalin, Ph.D., Department of Clinical Physiology, Karolinska Hospital M1:02, 171 76, Stockholm, Sweden.

	Abstract

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Phosphorylation of the -subunits of Na⁺,K⁺-adenosine triphosphatase in response to insulin, high extracellular glucose concentration, and phorbol 12-myristate 13-acetate was investigated in isolated rat soleus muscle. All three stimuli increased -subunit phosphorylation approximately 3-fold. Phorbol 12-myristate 13-acetate- and high glucose-induced phosphorylation of the -subunit was completely abolished by the PKC inhibitor GF109203X, whereas insulin-stimulated phosphorylation was only partially reduced. Notably, insulin stimulation resulted in phosphorylation of the -subunit on serine, threonine, and tyrosine residues, whereas high extracellular glucose or phorbol 12-myristate 13-acetate stimulation mediated phosphorylation only on serine and threonine residues. Insulin stimulation resulted in translocation of Na⁺,K⁺-adenosine triphosphatase ₂-subunit to the plasma membrane and increased Na⁺,K⁺-adenosine triphosphatase activity in the same membrane fraction. High glucose had no effect on -subunits distribution. Immunoprecipitation with antiphosphotyrosine antibody and subsequent Western blot analysis with anti-₁- and -₂-subunit antibodies revealed that both ₁- and ₂-subunit isoforms underwent phosphorylation on tyrosine residues in response to insulin, although with different time course and magnitude. Thus, we show that insulin-stimulated phosphorylation of Na⁺,K⁺-adenosine triphosphatase -subunit occurs via a PKC- and tyrosine kinase-dependent mechanism, whereas high glucose-induced phosphorylation is only PKC-dependent. Phosphorylation of Na⁺,K⁺-adenosine triphosphatase -subunits may be involved in regulation of Na⁺,K⁺-adenosine triphosphatase activity by insulin or high extracellular glucose in skeletal muscle.

	Introduction

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THE Na⁺,K⁺-ADENOSINE TRIPHOSPHATASE (NA⁺,K⁺- ATPase) is an integral membrane protein and is critically involved in maintenance of intracellular sodium and potassium concentrations, participating to the maintenance of cell volume and electrochemical gradients. In addition to these general functions, Na⁺,K⁺-ATPase promotes membrane repolarization and reuptake of extracellular potassium in excitable cells, including skeletal muscle (1). In view of its fundamental importance, the regulation of the Na⁺,K⁺-ATPase can be achieved by different mechanisms, including changes in intrinsic activity, subcellular distribution, and cellular abundance (2, 3, 4).

Short-term regulation of cellular Na⁺-pump activity can be achieved by phosphorylation of its -subunit (2, 3, 4, 5). Numerous studies have documented that the -subunit in Na⁺,K⁺-ATPase preparations purified from different species can be phosphorylated in vitro on serine and threonine residues by PKC, PKA, and PKG (6, 7, 8, 9). Activators of these protein kinases stimulate endogenous Na⁺,K⁺-ATPase -subunit phosphorylation in Xenopus oocyte homogenates (6, 9) and in intact cells (10, 11). However, the functional consequences of Na⁺,K⁺-ATPase -subunit phosphorylation are tissue- and protein kinase-specific (10, 11, 12, 13, 14, 15).

Skeletal muscle is one of the most important target tissues for insulin, a hormone which plays a major role in the control of both glucose transport and metabolism (16), as well as K⁺ uptake, and thereby controlling the plasma K⁺ concentration (17). This latter effect of insulin is likely to be achieved through a stimulation of Na⁺,K⁺-ATPase in skeletal muscle and is not secondary to an increase in [Na⁺]_i via Na⁺-H⁺ antiporter stimulation (18). Insulin participates in regulation of the sodium pump by phosphorylation. For instance, in kidney proximal convoluted tubules, insulin stimulates Na⁺,K⁺-ATPase activity in a PKC-independent manner. This effect is abolished by tyrosine kinase inhibition and mimicked by orthovanadate, a tyrosine phosphatase inhibitor (19). The insulin-stimulated increase in Na⁺,K⁺-ATPase activity has been reported to be dependent on the ₁-subunit phosphorylation at Tyr-10 in rat kidney proximal tubule cells (20). In contrast to the kidney proximal tubule (19, 21), the stimulation of Na-pump activity in muscle by insulin is PKC-dependent (22) and is mediated, in part, by an increase in cell surface appearance of Na⁺,K⁺-ATPase in rat skeletal muscle cells (23). After 30 min of in vivo insulin exposure, ₂- and ß₁-subunit abundance in the plasma membrane of rat muscle has been shown to be increased, with no change in ₁ or ß₂ distribution (24). Importantly, insulin-regulated glucose transporter GLUT4 and ₂-subunit of Na⁺,K⁺-ATPase, despite many similarities, do not share the same intramuscular location, suggesting that the signaling pathway mediating the translocation process might be different in each case (3). Recruitment of Na⁺,K⁺-ATPase molecules to the muscle plasma membrane has been observed in oxidative soleus and red gastrocnemius but not glycolytic white gastrocnemius muscle (24).

The major function of insulin is the control of whole-body glucose homeostasis. Elevated blood glucose level is the diagnostic characteristic of diabetes. Reduced Na⁺,K⁺-ATPase activity in a number of tissues has been reported in association with diabetes (25). Furthermore, high extracellular glucose concentrations have been shown to inhibit Na⁺,K⁺-ATPase activity in smooth muscle cells (26). Inhibition of Na⁺,K⁺-ATPase in pancreatic ß-cells, by high extracellular glucose, correlates with an increased degree of phosphorylation of the -subunit (27).

To date, the mechanisms of sodium pump activation and the role of protein kinase-mediated phosphorylation of Na⁺,K⁺- ATPase subunits, in response to insulin, have not been defined. We investigated whether insulin induces phosphorylation of the Na⁺,K⁺-ATPase in rat skeletal muscle and whether PKC and/or tyrosine kinases are involved in this process. In addition, we determined whether Na⁺,K⁺-ATPase phosphorylation, in response to insulin, is -subunit isoform-specific.

	Materials and Methods

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Antibodies and reagents
Immunoprecipitation of the Na⁺,K⁺-ATPase -subunit was performed using a polyclonal antibody, anti-NK1, raised against purified rat kidney holoenzyme (10, 20). Specific anti-₁-subunit monoclonal and anti-₂-subunit polyclonal antibodies (28) were generously provided by Dr. M. Caplan (Yale University, New Haven, CT). Antiphosphotyrosine PY20 antibody was from Transduction Laboratories, Inc. (Lexington, KY). Human insulin (Actrapid) was from Novo Nordisk AS (Copenhagen, Denmark). Phorbol 12-myristate 13-acetate (PMA), okadaic acid, HNPA-(AH)₃ (hydroxy-2-naphthalenylmethylphosphonic acid trisacetoxymethyl ester), peroxyvanadate, and GF109203X were from Calbiochem (La Jolla, CA). Dimethylsulfoxide from Calbiochem was used as a solvent for protein kinases and phosphatase inhibitors and activators. All other reagents were of analytical grade (Sigma, St. Louis, MO).

Animals and muscle incubation
Male Wistar rats (120–130 g) were purchased from B & K Universal (Sollentuna, Sweden) and housed in the animal facility at the Karolinska Hospital for 1 wk before use. The Animal Ethical Committee of the Karolinska Institute approved all protocols. Animals were fasted overnight before experiments. Rats were anesthetized with ip injection of sodium pentobarbital (5 mg/100 g BW). Soleus muscles were removed and split longitudinally. Media were prepared from pregassed (95% O₂-5% CO₂) Krebs Henseleit buffer (KHB) containing 5 mM HEPES and 0.1% BSA (RIA grade). Soleus muscles were incubated (30 min) in a shaking water bath (30 C) in 2 ml KHB, supplemented with 5 mM glucose and 15 mM mannitol. Muscles were transferred to fresh KHB and incubated without or with 120 nM insulin or 5 µM PMA. When pharmacological inhibitors were used, an additional 30-min incubation was introduced to preexpose the muscle to the inhibitor. Once added, inhibitors remained present for the duration of the experiment. The final concentration of dimethylsulfoxide was adjusted to 0.1% for each group. Incubation was terminated by freezing the muscle with tongs precooled in liquid nitrogen.

Incubation of rat soleus muscles with ³²Pi
Muscles were incubated for 60 min at 30 C in oxygenated low-phosphate KHB (0.3 mM Pi) supplemented with 5 mM glucose and 15 mM mannitol containing 5 mM HEPES and 0.1% BSA (RIA grade) to reduce the endogenous Pi content in the muscle. Muscles were then incubated in identical medium containing ³²Pi (1 mCi/ml) for 2 h. Protein kinase inhibitors, PMA, or insulin were added during the last 10–40 min of incubation time, as described above. Incubation was terminated by freezing muscles in liquid nitrogen. Muscles were homogenized as described below in Immunoprecipitation. The -subunit was immunoprecipitated, separated by SDS-PAGE, and transferred to nitrocellulose membranes. In every experiment, the amount of radioactivity incorporated into the -subunit was corrected for the amount of the protein detected by Western blot, and the quantitative data are shown as percent of basal.

Immunoprecipitation
Soleus muscles were pulverized in liquid nitrogen and homogenized in Eppendorf tubes with a Pellet Pestle Motor, (Kebo Lab, Stockholm, Sweden) in 0.5 ml ice-cold lysis buffer containing 20 mM Tris (pH 8.0), 135 mM NaCl, 1 mM MgCl₂, 2.7 mM KCl, 10 mM Na₄P₂O₇, 10 mM NaF, 1 mM Na₃VO₄, 1 µM okadaic acid, 1% Triton X-100, 10% vol/vol glycerol, 0.2 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin. Insoluble material was removed by centrifugation (12,000 x g for 10 min at 4 C). Protein was determined using a bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL). Aliquots of supernatant (750 µg protein) were immunoprecipitated overnight at 4 C with 50 µl of polyclonal anti-NK1 antibodies or antiphosphotyrosine antibody PY20. Immunoprecipitates were collected on protein A-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) and washed four times in lysis buffer; twice in 0.1 M Tris (pH 8.0) and 0.5 M LiCl; once in 10 mM Tris (pH 7.6), 0.15 M NaCl, and 1 mM EDTA; and once in 20 mM HEPES, 5 mM MgCl₂, and 1 mM dithiothreitol. Pellets were resuspended in Laemmli sample buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes and subjected to Western blot with appropriate antibodies. Phosphoproteins were analyzed using a PhosphoImager (Bio-Rad Laboratories, Inc., Hercules, CA), and quantitation was performed using the Molecular Imager software (Bio-Rad Laboratories, Inc.).

Phosphoamino acid analysis
The phosphorylated -subunit was immunoprecipitated and resolved by SDS-PAGE, and the ³²P-labeled Na⁺,K⁺-ATPase -subunits were identified on the membrane by PhosphoImager and cut out. Thereafter, the phosphorylated -subunit was hydrolyzed in 6 M HCl and analyzed by two-dimensional high-voltage electrophoresis on cellulose thin-layer plates. Phosphoamino acid analysis was performed essentially as described by Boyle et al. (29). Phosphoamino acids, on thin-layer electrophoresis plates, were analyzed using a PhosphoImager.

Subcellular fractionation of rat skeletal muscle membranes
Cell surface and intracellular membrane fractions were isolated by differential centrifugations and discontinuous sucrose gradients as previously described (23, 24). Rat soleus muscles were incubated with 120 nM insulin for 10 min and with 25 mM glucose for 30 min as described above. Approximately 350 mg rat skeletal muscle was weighed, minced, and initially homogenized with a Polytron at a low speed (setting 4, 2 x 10 sec) in homogenization buffer (20 mM Tris-HCl, 0.25 M sucrose, 1 mM EDTA, 1 µM okadaic acid, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin). The resulting homogenate was centrifuged for 10 min at 3,000 x g. The supernatant was collected and kept on ice. The pellet was resuspended in homogenization buffer and centrifuged again for 10 min at 3,000 x g. The two supernatants were pooled and centrifuged for 20 min at 12,000 x g. The supernatants were collected and then pelleted for 1 h at 150,000 x g. The crude membrane pellets were resuspended in 500 µl homogenization buffer and layered on top of a discontinuous density gradient consisting of 1.5 ml of 35%, 1.5 ml of 30%, and 1.5 ml of 25% sucrose. After centrifugation for 16 h at 77,000 x g, three protein fractions were collected: fraction 25 on top of the 25% layer; fraction 30 from the interphase 25–30%; fraction 35 from the interphase 30–35%. All the fractions were collected, diluted with 20 mM Tris-HCl (pH 7.4), and centrifuged for 60 min at 150,000 x g. Pellets were resuspended in 20 mM Tris-HCl, 0.25 M sucrose, pH 7.4. Protein concentrations were determined as described above. Fraction 25 has been previously characterized as a cell surface membrane fraction enriched with plasma membranes (23).

Determination of Na⁺,K⁺-ATPase activity
Na⁺,K⁺-ATPase activity was measured at maximum velocity (V_max) conditions in homogenates and membrane fractions, essentially as described (13, 14). Rat soleus muscles, frozen in liquid nitrogen, were homogenized in buffer (final vol, 300 µl) containing 50 mM Tris-HCl (pH 7.6), 2 mM EGTA, 250 mM sucrose, 1 µM okadaic acid, and 1 µM peroxyvanadate in glass/glass homogenizer. Homogenates were centrifuged at 12,000 x g for 10 min at 4 C, and supernatants were collected. Aliquots of the supernatants (protein content, 30–40 µg) or isolated membrane fractions (protein content, 2–3 µg) were transferred to the Na⁺,K⁺-ATPase assay medium (final vol, 100 µl), containing 50 mM NaCl, 5 mM KCl, 10 mM MgCl₂, 1mM EGTA, 50 mM Tris-HCl, 10 mM Na₂ATP, and -³²P-ATP (NEN Life Science Products; specific activity, 3000 Ci/mmol) in tracer amounts (3.3 nCi/µl), at 4 C. The samples were then incubated at 37 C for 15 min. The reaction was terminated by rapid cooling to 4 C and addition of a mixture of trichloroacetic acid/charcoal (5%/10%). After separating the charcoal phase (12,000 x g for 5 min) containing the nonhydrolyzed nucleotide, the ³²Pi liberated in the supernatant was counted. Na⁺,K⁺-ATPase activity was calculated as the difference between test samples (total ATPase activity) and samples assayed in a medium devoid of Na⁺ and K⁺ and in the presence of 2 mM ouabain (ouabain-insensitive ATPase activity). Protein determination was performed as described above.

Statistics
Comparisons between two experimental groups were made by the t test. For multiple comparisons, one-way ANOVA with Sheffe’s correction was used. P < 0.05 was considered significant.

	Results

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Phosphorylation of the -subunits of Na⁺,K⁺-ATPase from rat soleus muscle in response to insulin
To determine whether insulin promotes phosphorylation, Na⁺,K⁺-ATPase muscles were metabolically labeled with ³²Pi. Thereafter, muscles were either incubated with 120 nM insulin for 10 and 30 min, or 5 µM PMA, in the absence or presence of 5 µM of specific PKC inhibitor 2-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(-indol-3-yl)maleimide GF109203X (30, 31) for 30 min. Incubation of isolated rat soleus muscles in phosphorylation media in the presence of insulin led to phosphorylation of the Na⁺,K⁺-ATPase -subunit. Insulin increased the -subunit phosphorylation approximately 3-fold after 10 min of incubation (Fig. 1). Phosphorylation of the Na⁺,K⁺-ATPase -subunit was transient. Phosphorylation returned to basal levels after 30 min of insulin stimulation (Fig. 1), suggesting that, in this time period, the -subunit was dephosphorylated by protein phosphatases. PMA, a well-characterized PKC activator that potently induces Na⁺,K⁺-ATPase -subunit phosphorylation in a number of tissues (6, 10, 15), was used for comparison. PMA increased basal -subunit phosphorylation 3-fold. This PMA-dependent increase in phosphorylation was inhibited in the presence of 5 µM PKC inhibitor GF109203X.

View larger version (37K): [in this window] [in a new window]   Figure 1. Effect of insulin and PMA on the phosphorylation state of the immunoprecipitated Na+, K+-ATPase -subunit from rat soleus muscle. Isolated rat soleus muscles were metabolically labeled with 32Pi as described in Materials and Methods and incubated with 120 nM insulin for 10 and 30 min, or with 5 µM PMA in the absence or presence of 5 µM GF109203X (GFX) for 30 min. Muscles were homogenized, and equal amounts of protein were immunoprecipitated with anti-NK1 antibody. A representative autoradiogram is shown in the upper panel, and the quantitative data from four experiments (mean ± SE) are shown in the lower panel. *, P < 0.05 vs. basal.

Phosphorylation of the Na⁺,K⁺-ATPase -subunit in the absence or presence of protein kinase inhibitors

View larger version (39K): [in this window] [in a new window]   Figure 2. Phosphorylation of the Na+, K+-ATPase -subunit in the absence or presence of insulin, high extracellular glucose, and protein kinase inhibitors. Isolated rat soleus muscles were metabolically labeled with 32Pi as described in Materials and Methods and incubated with 120 nM insulin for 10 min, with 5 µM PMA for 30 min, or 25 mM glucose for 30 min in the absence or presence of 1 µM HNMPA-(AM)3, 1 or 5 µM GF109203X, as indicated in this figure and described in Materials and Methods. Na+, K+-ATPase -subunit was immunoprecipitated with anti-NK1 antibody. A representative autoradiogram is shown in the upper panel, and the quantitative data from four to seven experiments (mean ± SE) are shown in the lower panel. *, P < 0.05 vs. basal; , P < 0.05 vs. insulin-stimulated muscles.

Effect of high extracellular glucose on Na⁺,K⁺-ATPase -subunit phosphorylation
High extracellular glucose has been suggested to stimulate Na⁺,K⁺-ATPase -subunit phosphorylation by PKC-dependent mechanism in pancreatic ß-cells (27). High glucose concentrations induce PKCs translocation from cytosol to the membrane in rat soleus muscle (34). To assess whether high extracellular glucose induces Na-pump -subunit phosphorylation, we incubated rat soleus muscle in phosphorylation medium with 25 mM glucose. High glucose exposure was associated with a 3-fold increase in -subunit phosphorylation (Fig. 2). In contrast to insulin, the glucose-induced phosphorylation was abolished in the presence of 1 µM GF109203X, indicating that conventional and/or novel PKCs are involved in the process.

Phosphoamino acid analysis of Na⁺,K⁺-ATPase -subunit phosphorylated in rat soleus muscle
To identify the Na⁺,K⁺-ATPase -subunit amino acids phosphorylated in response to insulin, PMA, or high glucose concentration in rat skeletal muscle, a phosphoamino acid analysis of phosphorylated -subunit was performed. After stimulation by insulin, PMA, or high glucose concentrations, the Na⁺,K⁺-ATPase -subunit was primarily phosphorylated on serine residues (Fig. 3). A small amount of ³²P-phosphate was also detected on threonine residues. The same phosphoamino acid pattern has been reported previously after analysis of purified Na⁺,K⁺-ATPase phosphorylated by PKC in vitro (6, 7, 35). In contrast, a radiolabeled phosphotyrosine was detected only in the Na⁺,K⁺-ATPase -subunit immunoprecipitated from soleus muscle incubated with insulin.

View larger version (116K): [in this window] [in a new window]   Figure 3. Phosphoamino acid analysis of immunoprecipitated Na+, K+-ATPase -subunit phosphorylated in rat soleus muscle in response to insulin, PMA, or high extracellular glucose (25 mM). Muscles were treated as described for Fig. 2. Immunoprecipitated Na+, K+-ATPase -subunits were subjected to phosphoamino acid analysis as described in Materials and Methods. Representative autoradiograms of phosphoamino acids of immunoprecipitated -subunit phosphorylated in basal conditions in response to insulin, PMA, or high glucose concentration are shown. The circles represent the positions of unlabeled phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (P-Tyr). The additional spots represent the site of sample application (ori) and nonhydrolyzed peptides.

Insulin stimulated tyrosine phosphorylation of Na⁺,K⁺-ATPase -subunit isoforms in rat soleus muscle

View larger version (43K): [in this window] [in a new window]   Figure 4. Effect of insulin on the tyrosine phosphorylation state of the immunoprecipitated Na+, K+-ATPase different -subunit isoforms from rat soleus muscle. A, NK1 antibody recognizes both 1- and 2-isoforms of the Na+, K+-ATPase -subunit. Lysates from rat soleus muscles were prepared, and immunoprecipitation (IP) was performed with anti-NK1 antibody. Samples of muscle lysate (150 µg of protein), washed protein A pellet, and post-IP supernatant (150 µg of protein) were subjected to immunoblot with anti-1- and anti-2-subunit antibodies. B–D, Isolated rat soleus muscles were incubated with 120 nM insulin for the indicated amount of time. Muscles were homogenized as described in Materials and Methods, and equal amounts of protein were immunoprecipitated with PY20 antibody and subjected to immunoblot analysis. B, Representative immunoblot with anti-NK1 antibody (upper panel). Quantitative data from four experiments (mean ± SE) are shown in the lower panel. *, P < 0.05 vs. basal. C, Representative immunoblot with antibody against 1-subunit isoform. D, Representative immunoblot with antibody against 2-subunit isoform (upper panel). Quantitative data from four experiments (mean ± SE) are shown in the lower panel. *, P < 0.05 vs. basal.

Effect of insulin, PMA, and high extracellular glucose on Na⁺,K+-ATPase activity in rat soleus muscle
To assess whether insulin modified the intrinsic activity of the total Na⁺,K⁺-ATPase cellular pool, Na⁺,K⁺-ATPase activity was measured under V_max conditions in homogenates of soleus muscles previously incubated with insulin or PMA. To prevent possible dephosphorylation, the serine/threonine protein phosphatase inhibitor okadaic acid (1 µM) and the tyrosine phosphatase inhibitor peroxyvanadate (1 µM) were added to the homogenization medium. Okadaic acid and peroxyvanadate did not alter basal Na⁺,K⁺-ATPase activity (data not shown). Incubation with insulin did not significantly modify Na⁺,K⁺-ATPase activity (Fig. 5A). To investigate whether the lack of insulin effect is attributable to superimposition of an inhibitory effect of PKC-dependent phosphorylation and a stimulatory effect of PKC-independent phosphorylation, we incubated rat soleus muscles with the PKC inhibitor GF109203X and insulin. Preincubation with GF109203X did not unmask any effect of insulin on Na⁺,K⁺-ATPase activity. In contrast, a 30-min incubation with PMA led to 32% inhibition of Na⁺,K⁺-ATPase activity (Fig. 5B). To assess whether high extracellular glucose induces changes in Na⁺,K⁺-ATPase activity, we incubated rat soleus muscle in medium with 25 mM glucose. High glucose exposure led to a 30% decrease in Na⁺,K⁺-ATPase activity (Fig. 5B). The glucose-induced inhibition was abolished in the presence of both 1 and 5 µM GF109203X, indicating that this effect is PKC-dependent. The ouabain-insensitive ATPase activity did not change in response to insulin, PMA, or high extracellular glucose (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 5. Effect of insulin, PMA, and high extracellular glucose on the Na+, K+-ATPase activity in rat soleus muscle. Isolated muscles were incubated with 120 nM insulin for 10 and 30 min (A) or with 120 nM insulin for 10 min and 5 µM PMA or 25 mM glucose for 30 min in the absence or presence of 1 or 5 µM GF109203X (B) as indicated in figure and described in Materials and Methods. Muscle homogenates were assayed for Na+, K+-ATPase activity. Each bar represents the mean ± SE from 7–14 experiments performed in triplicate. *, P < 0.05 vs. basal.

Effect of insulin and high extracellular glucose on -subunits redistribution and Na⁺,K⁺-ATPase activity in cell surface membranes from rat soleus muscle

View larger version (37K): [in this window] [in a new window]   Figure 6. Changes in subcellular distribution of Na+, K+-ATPase 1- and 2-subunits and Na+, K+-ATPase activity in membrane fractions of rat soleus muscle after stimulation by insulin and high glucose. The abundance of GLUT1, GLUT4, and 1-, and 2-subunits of Na+, K+-ATPase was assayed in cell surface membrane fraction 25 from control and stimulated muscles. Representative autoradiograms are shown (A). The abundance of 2-subunit in cell surface membrane is shown as percentage of basal (B). Quantitative data from four to five experiments (mean ± SE) are shown. *, P < 0.05 vs. basal. Muscle crude membrane fractions (C) and cell surface membranes (D) were assayed for Na+, K+-ATPase activity. Each bar represents the mean ± SE from five to six experiments performed in triplicate. *, P < 0.05 vs. basal; n.s., not significant.

	Discussion

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A decrease in extracellular K⁺ concentration was one of the first reported actions of insulin (17). Skeletal muscle is a primary storage for dietary K⁺, and insulin plays a major role in removal of this ion from the circulation under physiological conditions, i.e. after a meal (25). Insulin-treated diabetic patients show an improved capacity for extra-renal clearance of an acute K⁺ load (37). These effects of insulin are likely to be achieved through a stimulation of Na⁺,K⁺-ATPase in skeletal muscle. In the present study, we found that insulin induces transient phosphorylation of the Na⁺,K⁺-ATPase -subunits in rat skeletal muscle (Fig. 1).

In contrast to results reported for kidney proximal convoluted tubule cells (20), insulin-stimulated Na⁺,K⁺-ATPase -subunit phosphorylation is partially PKC-dependent in rat soleus muscle. Insulin not only stimulates tyrosine phosphorylation, but also serine/threonine phosphorylation, of the Na⁺,K⁺-ATPase -subunit. Preincubation with 5 µM GF109203X, a specific PKC inhibitor, partially prevented the phosphorylation of the Na⁺,K⁺-ATPase -subunit. Interestingly, 1 µM GF109203X did not inhibit insulin-stimulated Na⁺,K⁺-ATPase -subunit phosphorylation, suggesting that atypical, rather then conventional/novel, PKC isoforms are likely involved. The insulin receptor tyrosine kinase inhibitor HNMPA-(AM)₃ completely blocked the insulin-mediated phosphorylation of the Na⁺,K⁺-ATPase -subunit. These results indicate that PKC activation is downstream of insulin receptor tyrosine kinase. Thus, insulin receptor stimulation may lead to an activation and translocation to the membrane of atypical PKC isoforms (38, 39), which may phosphorylate the Na⁺,K⁺-ATPase -subunits on serine and threonine residues.

To further compare the insulin-stimulated Na⁺,K⁺- ATPase -subunit phosphorylation with the PKC-mediated phosphorylation, we have used activators of PKC, specifically PMA, and high extracellular glucose concentrations that may be physiologically important for skeletal muscle. Indeed, both high extracellular glucose and insulin activate PKCs in skeletal muscle (22, 34). High glucose concentrations induce translocation of PKCß2 and (to a lesser extent) PKC, to the membrane in rat soleus muscle (34). Hyperglycemia is known to reduce Na⁺,K⁺-ATPase activity via a PKC-dependent mechanism in vascular smooth muscle cells (26) and in pancreatic ß-cells (27). Our results from the phosphorylation experiments and the phosphoamino acid analysis provide direct evidence of PKC-dependent Na-pump phosphorylation in response to an increase in the extracellular glucose concentration in skeletal muscle, a tissue which accounts for 80% of total glucose disposal. In contrast to insulin-induced phosphorylation, high glucose most likely activates different PKC isoforms in skeletal muscle (in particular, conventional and novel), the activity of which is regulated by [Ca²⁺]_i and diacylglycerol. The specific role of different PKC isoforms in phosphorylation and regulation of skeletal muscle Na-pump warrants further investigation.

Interestingly, we show threonine phosphorylation of Na⁺,K⁺-ATPase -subunit in skeletal muscle in response to insulin. PKC-mediated phosphorylation of Thr-15 has been described in a detailed site-directed mutagenesis study of ₁-subunit from Bufo marinus, although Thr-15 accounted for only a small percentage of total phosphorylation (40, 41). PKC-mediated phosphorylation on threonine residues has also been described in several other studies, including in vitro phosphorylation of Na⁺,K⁺-ATPase preparations from different species containing the ₁-subunit (6, 7, 35) and intact rat kidney proximal convoluted tubule cells (20). Thr-15 is not present in rat ₁-subunit, although rat ₂- has Thr at positions 14 and 15 (41). Furthermore, PKC can induce phosphorylation on only threonine residues in a fusion protein containing the N-terminus of rat ₂-subunit (41). The present work demonstrates that threonine residues are phosphorylated under different stimulation conditions in the Na⁺,K⁺-ATPase -subunit in rat soleus muscle. Regardless of whether threonine residues are a target for PKC or for another serine/threonine protein kinase, the sites of phosphorylation and their physiological role(s) remain to be determined.

Determination of the total phosphorylation level of the Na⁺,K⁺-ATPase with ³²P-labeling does not allow the detection of isoform-specific phosphorylation, because the antibody used recognizes both ₁- and ₂-subunit isoforms (Fig. 4A). Furthermore, a specific immunoprecipitating anti-₂-subunit isoform antibody was not available. However, immunoprecipitation with antiphosphotyrosine antibody and subsequent Western blot analysis with anti-₁- and -₂- subunit antibodies revealed that both ₁- and ₂-subunit isoforms underwent phosphorylation on tyrosine residues in response to insulin in rat soleus muscle, although with a different time course (Fig. 4, C–D). Thus, phosphorylation of the ₂-, but not the ₁-, subunit isoform seems to account for the observed insulin effect.

The molecular nature of the tyrosine kinase that phosphorylates Na⁺,K⁺-ATPase in soleus muscle is not yet identified. Although the insulin receptor tyrosine kinase inhibitor HNMPA-(AM)₃ completely blocked insulin-mediated phosphorylation of the Na⁺,K⁺-ATPase -subunit, involvement of nonreceptor tyrosine kinases cannot be excluded (42). Moreover, recent studies reveal a functional association between the insulin receptor and downstream effectors, including phosphatidylinositol 3-kinase and nonreceptor tyrosine kinase c-src (43). Our preliminary results indicate that c-src phosphorylates the -subunit of the Na⁺,K⁺-ATPase in vitro in skeletal muscle plasma membrane preparation (Chibalin, A. V., M. V. Kovalenko, and J. R. Zierath, unpublished results).

Although Tyr-10 is a target for tyrosine kinases in the rat Na⁺,K⁺-ATPase ₁-subunit isoform (20), the same residue is not likely to undergo phosphorylation in ₂-subunit isoform. Tyr-10 is conserved and present in the rat ₂-subunit sequence; however, the environment of this residue is different. In the rat ₁-subunit isoform, Tyr-10 is located in the sequence –DKYEP- and surrounded by negatively charged amino acid residues at positions -2 and +1. This characterizes a consensus phosphorylation site for nonreceptor tyrosine kinases (44). In the rat ₂-subunit isoform, the tyrosine residue is located in the sequence –REYSP-, and it is surrounded by only one negatively charged residue at position -1. Although we have not yet attempted to localize the tyrosine phosphorylation site in the rat ₂-subunit sequence experimentally, sequence comparison with PhosphoBase database (The Center for Biological Sequence Analysis, The Technical University of Denmark, Lyngly, Denmark) (45, 46) predicts Tyr-543 as a most probable target for tyrosine kinases in rat Na⁺,K⁺-ATPase ₂-subunit. This tyrosine residue is located in the large cytoplasmic loop of Na⁺,K⁺-ATPase -subunit (5), and it is surrounded by the sequence –AYMELG-. Interestingly, the sequence motif –YMEL- is very similar to clathrin adaptor protein 2-recognized tyrosine-based endocytic motif (47), used for sorting proteins from the plasma membrane to endosomes (48), including ₁-subunit of rat Na⁺,K⁺-ATPase (49). An attractive hypothesis could be that insulin-stimulated phosphorylation of Tyr-543 arrests the formation of an endocytic complex of Na⁺,K⁺-ATPase ₂-subunit, adaptor protein 2, and clathrin. This may prevent the Na⁺,K⁺-ATPase ₂-subunit from undergoing endocytosis from the plasma membrane and may lead to an increased ₂-subunit abundance in plasma membrane caused by constitutive exocytosis. This hypothesis may offer a partial explanation for the mechanism involved in insulin-stimulated Na⁺,K⁺-ATPase ₂-subunit translocation to the plasma membrane in skeletal muscle. However, the involvement of PKC-dependent phosphorylation in regulation of the Na⁺,K⁺-ATPase intracellular traffic cannot be excluded (13, 14). The transient time course of ₂-subunit phosphorylation would prevent hyperactivity of the Na⁺,K⁺-ATPase in a plasma membrane that can lead to hyperpolarization of the membrane and hypokalemea. This transient phosphorylation may be attributable to protein phosphatase activity. Serine/threonine protein phosphatases are involved in insulin regulation of Na⁺,K⁺-ATPase activity in cultured L6 rat skeletal muscle cells (50). However, the involvement of specific phosphatases in the regulation of Na⁺,K⁺-ATPase activity by insulin in intact muscle is difficult to prove definitively without detailed knowledge regarding the multiple phosphorylation sites on the -subunit isoforms.

Stimulation of soleus muscle by insulin does not significantly change total cellular Na⁺,K⁺-ATPase catalytic activity measured at V_max in muscle homogenates. Insulin has been reported to stimulate ouabain-suppressible Rb⁺ uptake in isolated rat soleus muscle (18) and ₂-subunit translocation to muscle surface membranes after insulin injection of animals (23, 24). We have shown that ex vivo incubation of isolated rat soleus muscle with insulin leads to ₂-subunit translocation to cell surface membranes, which correlates with an increase in total Na⁺,K⁺-ATPase activity. Importantly, the increase in Na⁺,K⁺-ATPase activity can be observed only in plasma membrane-enriched, but not in crude, membrane fractions. The majority (75–80%) of the Na⁺,K⁺-ATPase pool in rat soleus muscle corresponds to the ₂-subunit (51); therefore, comparison of ₂-subunit abundance and Na⁺,K⁺-ATPase activity in cell surface membranes fractions suggests that the observed increase in activity is a result of Na⁺,K⁺-ATPase translocation to the plasma membrane. Taken together, the data support the hypothesis that insulin stimulation of Na⁺,K⁺-ATPase in skeletal muscle is attributable to an increase in the number of Na-pump units in the plasma membrane, rather than a change in the activity of a single pump unit. However, a possible change in Na⁺ affinity of the enzyme cannot be excluded.

In contrast to insulin, muscle exposure to PMA or high extracellular glucose leads to decrease in Na⁺,K⁺-ATPase activity measured in homogenates. The inhibition of Na⁺,K⁺-ATPase activity by high glucose is PKC-dependent. Interestingly, the inhibitory effect is maximal in homogenates and decreases throughout membrane purification. This could be explained as a result of dephosphorylation of the Na⁺,K⁺-ATPase during the long process of membrane fractionation. Furthermore, proteins which can bind to phosphorylated Na⁺,K⁺-ATPase -subunit and may inhibit the ATPase activity of sodium pump units may be washed away during series of centrifugations.

In conclusion, the present study demonstrates that insulin and high extracellular glucose phosphorylate Na⁺,K⁺-ATPase -subunit in skeletal muscle at multiple sites. High extracellular glucose promotes phosphorylation on only serine and threonine residues, whereas insulin stimulates phosphorylation on both serine/threonine and tyrosine residues. Although the phosphorylation state needs to be directly linked with changes in Na-pump activity in skeletal muscle, this mechanism is likely to play an important role maintaining the intracellular distribution of Na⁺,K⁺-ATPase units and the regulation of Na⁺- and K⁺-gradients.

	Acknowledgments

 
We would like to thank Dr. M. Caplan for the kind gift of anti-₁- and anti- ₂-subunit antibodies. We especially thank Dr. Arne Östman and Dr. Anna Krook for helpful discussions and critical reading of the manuscript. We also thank Dr. Jorge Rincón and Maria Fernström for their contributions to the muscle incubation experiments.

	Footnotes

 
This work was supported by grants from the Swedish Medical Research Council, the Swedish Diabetes Association, Foundation for Strategic Research, the Novo-Nordisk Foundation, Thurings Foundation, Tore Nilsons Foundation, Åke Wiberg Foundation, Lars Hiertas Memorial Foundation (to A.C., J.R.Z., and H.W.-H.), and the Swiss National Science Foundation Grant 31-50643.97 (to E.F.).

Abbreviations: ATPase, Adenosine triphosphatase; GLUT, glucose transporter; HNMPA-(AM)₃, hydroxy-2-naphthalenylmethylphosphonic acid trisacetoxymethyl ester; KHB, Krebs Henseleit buffer; PMA, phorbol 12-myristate 13-acetate; V_max, maximum velocity.

Received November 13, 2000.

Accepted for publication April 4, 2001.

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